Role of sodium and potassium ions in regulation of glucose metabolism in cultured astroglia.

Effects of increasing extracellular K+ or intracellular Na+ concentrations on glucose metabolism in cultures of rat astroglia and neurons were examined. Cells were incubated in bicarbonate buffer, pH 7.2, containing 2 mM glucose, tracer amounts of [14C]deoxyglucose ([14C]dGlc), and 5.4, 28, or 56 mM KCl for 10, 15, or 30 min, and then for 5 min in [14C]dGlc-free buffer to allow efflux of unmetabolized [14C]dGlc. Cells were then digested and assayed for labeled products, which were shown to consist of 96-98% [14C]deoxyglucose 6-phosphate. Increased K+ concentrations significantly raised [14C]deoxyglucose 6-phosphate accumulation in both neuronal and mixed neuronal-astroglial cultures at 15 and 30 min but did not raise it in astroglial cultures. Veratridine (75 microM), which opens voltage-dependent Na+ channels, significantly raised rates of [14C]dGlc phosphorylation in astroglial cultures (+20%), and these elevations were blocked by either 1 mM ouabain, a specific inhibitor of Na+,K(+)-ATPase (EC 3.6.1.37), or 10 microM tetrodotoxin, which blocks Na+ channels. The carboxylic sodium ionophore, monensin (10 microM), more than doubled [14C]dGlc phosphorylation; this effect was only partially blocked by ouabain and unaffected by tetrodotoxin. L-Glutamate (500 microM) also stimulated [14C]dGlc phosphorylation in astroglia--not through N-methyl-D-aspartate or non-N-methyl-D-aspartate receptor mechanisms but via a Na(+)-dependent glutamate-uptake system. These results indicate that increased uptake of Na+ can stimulate energy metabolism in astroglial cells.

Crystallographic evidence for the action of potassium, thallium, and lithium ions on fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase (Fru-1,6-Pase; D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) requires two divalent metal ions to hydrolyze alpha-D-fructose 1,6-bisphosphate. Although not required for catalysis, monovalent cations modify the enzyme activity; K+ and Tl+ ions are activators, whereas Li+ ions are inhibitors. Their mechanisms of action are still unknown. We report here crystallographic structures of pig kidney Fru-1,6-Pase complexed with K+, Tl+, or both Tl+ and Li+. In the T form Fru-1,6-Pase complexed with the substrate analogue 2,5-anhydro-D-glucitol 1,6-bisphosphate (AhG-1,6-P2) and Tl+ or K+ ions, three Tl+ or K+ binding sites are found. Site 1 is defined by Glu-97, Asp-118, Asp-121, Glu-280, and a 1-phosphate oxygen of AhG-1,6-P2; site 2 is defined by Glu-97, Glu-98, Asp-118, and Leu-120. Finally, site 3 is defined by Arg-276, Glu-280, and the 1-phosphate group of AhG-1,6-P2. The Tl+ or K+ ions at sites 1 and 2 are very close to the positions previously identified for the divalent metal ions. Site 3 is specific to K+ or Tl+. In the divalent metal ion complexes, site 3 is occupied by the guanidinium group of Arg-276. These observations suggest that Tl+ or K+ ions can substitute for Arg-276 in the active site and polarize the 1-phosphate group, thus facilitating nucleophilic attack on the phosphorus center. In the T form complexed with both Tl+ and Li+ ions, Li+ replaces Tl+ at metal site 1. Inhibition by lithium very likely occurs as it binds to this site, thus retarding turnover or phosphate release. The present study provides a structural basis for a similar mechanism of inhibition for inositol monophosphatase, one of the potential targets of lithium ions in the treatment of manic depression.

Evidence for the existence of a sulfonylurea-receptor-like protein in plants: Modulation of stomatal movements and guard cell potassium channels by sulfonylureas and potassium channel openers

Limitation of water loss and control of gas exchange is accomplished in plant leaves via stomatal guard cells. Stomata open in response to light when an increase in guard cell turgor is triggered by ions and water influx across the plasma membrane. Recent evidence demonstrating the existence of ATP-binding cassette proteins in plants led us to analyze the effect of compounds known for their ability to modulate ATP-sensitive potassium channels (K-ATP) in animal cells. By using epidermal strip bioassays and whole-cell patch-clamp experiments with Vicia faba guard cell protoplasts, we describe a pharmacological profile that is specific for the outward K+ channel and very similar to the one described for ATP-sensitive potassium channels in mammalian cells. Tolbutamide and glibenclamide induced stomatal opening in bioassays and in patch-clamp experiments, a specific inhibition of the outward K+ channel by these compounds was observed. Conversely, application of potassium channel openers such as cromakalim or RP49356 triggered stomatal closure. An apparent competition between sulfonylureas and potassium channel openers occurred in bioassays, and outward potassium currents, previously inhibited by glibenclamide, were partially recovered after application of cromakalim. By using an expressed sequence tag clone from an Arabidopsis thaliana homologue of the sulfonylurea receptor, a 7-kb transcript was detected by Northern blot analysis in guard cells and other tissues. Beside the molecular evidence recently obtained for the expression of ATP-binding cassette protein transcripts in plants, these results give pharmacological support to the presence of a sulfonylurea-receptor-like protein in the guard-cell plasma membrane tightly involved in the outward potassium channel regulation during stomatal movements.

A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells1

Inactivation of inward-rectifying K+ channels (IK,in) by a rise in cytosolic free [Ca2+] ([Ca2+]i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca2+]i action on IK,in has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca2+]i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778–4783) while recording IK,in under a voltage clamp and [Ca2+]i by Fura-2 fluorescence ratiophotometry. IK,in inactivation correlated positively with [Ca2+]i and indicated a Ki of 329 ± 31 nm with cooperative binding of four Ca2+ ions per channel. IK,in was promoted by the Ca2+ channel antagonists Gd3+ and calcicludine, both of which suppressed the [Ca2+]i rise, but the [Ca2+]i rise was unaffected by the K+ channel blocker Cs+. We also found that ryanodine, an antagonist of intracellular Ca2+ channels that mediate Ca2+-induced Ca2+ release, blocked the [Ca2+]i rise, and Mn2+ quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca2+]i control of IK,in and to roles for discrete Ca2+ flux pathways in feedback control of the K+ channels by membrane voltage.

Calculated 11B–13C NMR chemical shift relationship in hypercoordinate methonium and boronium ions

The boronium-carbonium continuum was extended to include hypercoordinated protonated methanes and their boron analogs. The 11B NMR chemical shifts of the hypercoordinated hydriodo boron compounds and the 13C NMR chemical shifts of the corresponding isoelectronic and isostructural carbocations were calculated by using the GIAO-MP2 method. The data show good linear correlation between 11B and 13C NMR chemical shifts, which indicates that the same factors that determine the chemical shifts of the boron nuclei also govern the chemical shifts of carbon nuclei of these hypercoordinated hydriodo compounds.

Cyclic AMP regulates potassium channel expression in C6 glioma by destabilizing Kv1.1 mRNA

The tissue distributions and physiological properties of a variety of cloned voltage-gated potassium channel genes have been characterized extensively, yet relatively little is known about the mechanisms controlling expression of these genes. Here, we report studies on the regulation of Kv1.1 expressed endogenously in the C6 glioma cell line. We demonstrate that elevation of intracellular cAMP leads to the accelerated degradation of Kv1.1 RNA. The cAMP-induced decrease in Kv1.1 RNA is followed by a decrease in Kv1.1 protein and a decrease in the whole cell sustained K+ current amplitude. Dendrotoxin-I, a relatively specific blocker of Kv1.1, blocks 96% of the sustained K+ current in glioma cells, causing a shift in the resting membrane potential from −40 mV to −7 mV. These data suggest that expression of Kv1.1 contributes to setting the resting membrane potential in undifferentiated glioma cells. We therefore suggest that receptor-mediated elevation of cAMP reduces outward K+ current density by acting at the translational level to destabilize Kv1.1 RNA, an additional mechanism for regulating potassium channel gene expression.

Three metal ions at the active site of the Tetrahymena group I ribozyme

Metal ions are critical for catalysis by many RNA and protein enzymes. To understand how these enzymes use metal ions for catalysis, it is crucial to determine how many metal ions are positioned at the active site. We report here an approach, combining atomic mutagenesis with quantitative determination of metal ion affinities, that allows individual metal ions to be distinguished. Using this approach, we show that at the active site of the Tetrahymena group I ribozyme the previously identified metal ion interactions with three substrate atoms, the 3′-oxygen of the oligonucleotide substrate and the 3′- and 2′-moieties of the guanosine nucleophile, are mediated by three distinct metal ions. This approach provides a general tool for distinguishing active site metal ions and allows the properties and roles of individual metal ions to be probed, even within the sea of metal ions bound to RNA.

Modulation of potassium channel function by methionine oxidation and reduction

Oxidation of amino acid residues in proteins can be caused by a variety of oxidizing agents normally produced by cells. The oxidation of methionine in proteins to methionine sulfoxide is implicated in aging as well as in pathological conditions, and it is a reversible reaction mediated by a ubiquitous enzyme, peptide methionine sulfoxide reductase. The reversibility of methionine oxidation suggests that it could act as a cellular regulatory mechanism although no such in vivo activity has been demonstrated. We show here that oxidation of a methionine residue in a voltage-dependent potassium channel modulates its inactivation. When this methionine residue is oxidized to methionine sulfoxide, the inactivation is disrupted, and it is reversed by coexpression with peptide methionine sulfoxide reductase. The results suggest that oxidation and reduction of methionine could play a dynamic role in the cellular signal transduction process in a variety of systems.

hSK4, a member of a novel subfamily of calcium-activated potassium channels

The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca2+ (EC50 of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.

A human intermediate conductance calcium-activated potassium channel

An intermediate conductance calcium-activated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is ≈50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K0.5 = 0.3 μM). Although the K0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3.5). Single-channel current amplitudes reflected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (Ki = 2.5 nM) and clotrimazole (Ki = 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 μM). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel.

Ceramide-induced inhibition of T lymphocyte voltage-gated potassium channel is mediated by tyrosine kinases

The n-type K+ channel (n-K+, Kv1.3) in lymphocytes has been recently implicated in the regulation of Fas-induced programmed cell death. Here, we demonstrate that ceramide, a lipid metabolite synthesized upon Fas receptor ligation, inhibits n-K+ channel activity and induces a tyrosine phosphorylation of the Kv1.3 protein in Jurkat T lymphocytes. Tyrosine phosphorylation of the n-K+ channel correlated with an activation of the Src-like tyrosine kinase p56lck upon cellular treatment with the ceramide analog C6-ceramide. Because genetic deficiency of p56lck or inhibition of Src-like tyrosine kinases by herbimycin A prevented ceramide-mediated n-K+ channel inhibition and tyrosine phosphorylation, we propose a ceramide-initiated activation of p56lck resulting in tyrosine phosphorylation and inhibition of the n-K+ channel protein.

ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae

A K+ channel gene has been cloned from Drosophila melanogaster by complementation in Saccharomyces cerevisiae cells defective for K+ uptake. Naturally expressed in the neuromuscular tissues of adult flies, this gene confers K+ transport capacity on yeast cells when heterologously expressed. In Xenopus laevis oocytes, expression yields an ungated K+-selective current whose attributes resemble the “leak” conductance thought to mediate the resting potential of vertebrate myelinated neurons but whose molecular nature has long remained elusive. The predicted protein has two pore (P) domains and four membrane-spanning helices and is a member of a newly recognized K+ channel family. Expression of the channel in flies and yeast cells makes feasible studies of structure and in vivo function using genetic approaches that are not possible in higher animals.

Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line. Although this recombinant channel shares many functional properties with the “small-conductance” basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by conventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polarized renal epithelial cell line. Either the 11-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6-amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3. As determined by patch-clamp and two-microelectrode voltage-clamp analyses in Xenopus oocytes, neither tag affected channel function; no differences in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and the tagged constructs. MDCK cells were transfected with tagged CCD-IRK3, and several stable clonal cell lines were generated by neomycin-resistance selection. Immunoprecipitation studies with anti-P5D4 or anti-AU1 antibodies readily detected the predicted-size 50-kDa protein in the transfected cells lines but not in wild-type or vector-only (PcB6) transfected MDCK cells. As visualized by indirect immunofluorescence and confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane. To assure that the VSV G tag was not responsible for the targeting, the P5D4 epitope modified by a site-directed mutagenesis (Y2F) to remove a potential basolateral targeting signal contained in this tag. VSV(Y2F) was also detected exclusively on the basolateral membrane, confirming bona fide IRK3 basolateral expression. These observations, with our functional studies, suggest that CCD-IRK3 may encode the small-conductance CCD basolateral K channel.

Scanning mutagenesis of the putative transmembrane segments of Kir2.1, an inward rectifier potassium channel

Structural models of inward rectifier K+ channels incorporate four identical or homologous subunits, each of which has two hydrophobic segments (M1 and M2) which are predicted to span the membrane as α helices. Since hydrophobic interactions between proteins and membrane lipids are thought to be generally of a nonspecific nature, we attempted to identify lipid-contacting residues in Kir2.1 as those which tolerate mutation to tryptophan, which has a large hydrophobic side chain. Tolerated mutations were defined as those which produced measurable inwardly rectifying currents in Xenopus oocytes. To distinguish between water-accessible positions and positions adjacent to membrane lipids or within the protein interior we also mutated residues in M1 and M2 individually to aspartate, since an amino acid with a charged side chain should not be tolerated at lipid-facing or interior positions, due to the energy cost of burying a charge in a hydrophobic environment. Surprisingly, 17 out of 20 and 17 out of 22 non-tryptophan residues in M1 and M2, respectively, tolerated being mutated to tryptophan. Moreover, aspartate was tolerated at 15 out of 22 and 15 out of 21 non-aspartate M1 and M2 positions respectively. Periodicity in the pattern of tolerated vs. nontolerated mutations consistent with α helices or β strands did not emerge convincingly from these data. We consider the possibility that parts of M1 and M2 may be in contact with water.

Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism

In the COS7 cells transfected with cDNAs of the Kir6.2, SUR2A, and M1 muscarinic receptors, we activated the ATP-sensitive potassium (KATP) channel with a K+ channel opener and recorded the whole-cell KATP current. The KATP current was reversibly inhibited by the stimulation of the M1 receptor, which is linked to phospholipase C (PLC) by the Gq protein. The receptor-mediated inhibition was observed even when protein kinase C (PKC) was inhibited by H-7 or by chelating intracellular Ca2+ with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA) included in the pipette solution. However, the receptor-mediated inhibition was blocked by U-73122, a PLC inhibitor. M1-receptor stimulation failed to inhibit the KATP current activated by the injection of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) through the whole-cell patch pipette. The receptor-mediated inhibition became irreversible when the replenishment of PIP2 was blocked by wortmannin (an inhibitor of phosphatidylinositol kinases), or by including adenosine 5′-[β,γ–imido]triphosphate (AMPPNP, a nonhydrolyzable ATP analogue) in the pipette solution. In inside-out patch experiments, the ATP sensitivity of the KATP channel was significantly higher when the M1 receptor in the patch membrane was stimulated by acetylcholine. The stimulatory effect of pinacidil was also attenuated under this condition. We postulate that stimulation of PLC-linked receptors inhibited the KATP channel by increasing the ATP sensitivity, not through PKC activation, but most probably through changing PIP2 levels.